Hello, I'm trying to get this technique to work on K ion channel subunits in murine 4% PFA fixed spinal cord slices that have been treated with Na Citrate for 30mins at 70 degrees and blocked for an hour with donkey serum. Primary antibodies are raised in mouse and rabbit and work well in normal IHC and what we expected to work well as a positive control is not working either-has anyone got any suggestions or had issues with this kit before?
Hi, not sure I can be of much help as I haven't used PLA on tissue slices, but just would like to join the discussion. Have you tried increasing the concentration of the two primary antibodies and/or playing with the duration of the amplification step?
I used PLA kit on mono-layer cell culture fixed with methanol, and we are having problems with the negative control instead - we detected massive signals between a protein on the extracellular side of the plasma membrane and a cytoplasmic protein (but of course methanol fixation has dissolved the membrane so maybe this is not too much of a surprise...).
Hello, We used Duolink on FFPE meningioma with quite good results. Comparing to your protocol, antigen retrieval was perfomed at higher temperature (97°C, 20 min). We also used triton to increase permeability.
Best
GG
I have been using DuoLink PLA in monkey fixed tissue for a long time. Please see enclosed paper. Best, Jose
Thank you very much for your suggestions, I'll modify the protocol and keep optimising it!
Best
Pierce
Dear Pierce,
Many thanks for your reply. If you have any further question, please feel free to contact me at any time.
All the best and have a nice day, Jose L.
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